Search PubMed⌕ Search

Biomedical subjects

M Ward

Publications and source records attributed to M Ward.

At least 19 recordsLinked to original sources
PubMed1992

Transfer and expression of the human multidrug resistance gene in mouse erythroleukemia cells.

Gene therapy in humans requires the transplantation of genetically modified cells, and it is important to select only those cells capable of expressing high levels of protein from the transferred gene. Expression of the human multiple drug resistance (MDR) gene confers resistance to a variety of compounds in vitro and in vivo. To determine the feasibility of conferring recipient erythroid cells with the MDR phenotype, we have transduced mouse erythroleukemia cells (MELC) with the MDR gene in a retroviral vector. We show here that MELC clones resistant to exposure to colchicine (an MDR-responsive agent) can be isolated, and demonstrate high levels of MDR RNA and protein expression. Increasing doses of colchicine increase the level of MDR RNA and protein expression significantly. These results indicate that it is possible to transfer and express the human MDR phenotype in mouse erythroid cells by retrovirally mediated gene transfer, and that drug selection can be used to enrich or purify populations of cells containing and expressing this gene.

3T3 Cells↗
PubMed1992

Transfer and expression of the human multiple drug resistance gene into live mice.

The human multiple drug resistance (MDR) gene has been used as a selectable marker to increase the proportion of bone marrow cells that contain and express this gene by drug selection. By constructing retroviral vectors containing and expressing the MDR gene and a nonselectable gene such as the beta-globin gene, enrichment for cells containing both of these genes can be achieved. A retroviral construct containing MDR cDNA in a Harvey virus-based vector has been used to transfect our ecotropic 3T3 retroviral packaging line GP+E86. Clones have been isolated by exposure of the retrovirally transfected cells (MDR producer cells) to colchicine (60 ng/ml), a selective agent that kills MDR-negative cells. Flow cytometry analysis (fluorescence-activated cell sorting) with an antibody to MDR demonstrates expression of human MDR protein on the surface of these colchicine-resistant producer clones. Untransfected GP+E86 cells are negative. Colchicine-resistant clones were titered using clone supernatants and the highest titer clone (4 x 10(4) viral particles per ml) was cocultured with 10(6) donor mouse bone marrow cells for 24-48 hr. The donor cells were then injected into congenic irradiated mice, and the presence of the MDR gene was assayed by the polymerase chain reaction (PCR) analysis using MDR-specific primers. In one experiment eight of nine transduced mice were positive for MDR by PCR of peripheral blood 14 and 50 days posttransplantation; after 240 days three of nine transduced mice were positive. Bone marrow obtained from one of these positive animals was stained with the MDR monoclonal antibody and the granulocyte population was analyzed by FACS. Approximately 14% of the total granulocyte pool contain increased levels of MDR protein. In addition, the bone marrow cells of several mice initially positive for MDR gene by PCR, and subsequently negative, were exposed to taxol, a drug whose detoxification depends on MDR gene expression; a positive signal was obtained in all of these mice, indicating drug selection of MDR-positive marrow cells. Cell sorting studies of these mice also show an increased number of high-MDR-expressing marrow cells, selected after exposure to taxol. Thus, in this live animal model MDR transduction is effective in selecting a human MDR-expressing population of marrow cells resistant to taxol chemotherapy. This strategy may, thus, be useful in humans to prevent the marrow toxicity induced by anticancer agents such as taxol and as a selectable marker to enrich for cells simultaneously transduced with a nonselectable gene.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1992

Loss of heterozygosity at the human RAP1A/Krev-1 locus is a rare event in colorectal tumors.

Kirsten-ras-revertant-1 (Krev-1/Rap1A) is a recently identified tumor suppressor gene which induces flat revertants when introduced into a variety of ras-transformed cell lines in vitro. Since 47% of colorectal carcinomas have transforming mutations in ras protooncogenes, and since Krev-1 is expressed at high levels in normal colonic mucosa, we hypothesized that inactivation at the Krev-1 locus may be necessary for transformation of colonic cells. Loss of heterozygosity is a common method of inactivation of tumor suppressor genes in colorectal tumors. Therefore, we analyzed loss of heterozygosity in 52 patients with sporadic colorectal cancer. Because Krev-1 had no previously described polymorphisms, we first identified a BclI restriction fragment length polymorphism which showed 40% heterozygosity in 50 unrelated individuals. However, only one tumor from 18 informative patients showed allelic loss at the Krev-1 locus. This suggests that loss of heterozygosity is not a common mechanism of inactivation at the Krev-1 locus in colorectal cancer. However, the results do not exclude a role for Krev-1 in the etiology of this neoplasm because inactivation may occur by other mechanisms.

Alkaline Phosphatase↗
PubMed1992

The power of a place: opening the college classroom to people with serious mental illness.

Presented the structure and implementation of a university-based practicum course on social relationships for people with serious mental illness and college undergraduates. Grounded in an ecological view of social settings, cooperative learning models of education and mutual help principles, the practicum was designed to create a collaborative classroom setting where undergraduates and people with serious mental illness could both develop and enhance their own interpersonal skills and social network ties. The practicum demonstrates how a university can use its resources to help address community needs while simultaneously enhancing its mission of teaching and research. The role of social context in creating collaborative relationships among participants and the use of the university as a community resource are discussed.

Adaptation, Psychological↗
PubMed1992

Heterotypic protection of mice against chlamydial salpingitis and colonization of the lower genital tract with a human serovar F isolate of Chlamydia trachomatis by prior immunization with recombinant serovar L1 major outer-membrane protein.

Intrauterine infection of mice with a human genital tract isolate of Chlamydia trachomatis (serovar F) resulted in salpingitis. In some cases, oviduct damage was sufficient to cause infertility due to lumenal blockage. Parenteral immunization with a purified, heterologous, recombinant major outer-membrane (rMOMP) preparation reduced the proportion of animals developing severe salpingitis by 77% compared with mock-immunized controls, but failed to reduce chlamydial colonization of the lower genital tract. In contrast, mice immunized with rMOMP directly into the Peyer's patches to stimulate mucosal immunity shed fewer chlamydiae from the vagina than controls, but showed little reduction in oviduct damage. No consistent correlation was observed between antibody levels to rMOMP in immunized mice and reduced lower genital tract colonization. Immunization with rMOMP via the presacral space, a route previously shown to stimulate mucosal immunity in the genital tract, produced high levels of circulating anti-rMOMP IgG but only traces of anti-rMOMP IgA in vaginal secretions. There was no difference in the severity of salpingitis in these animals compared with mock-immunized controls. Immunization with rMOMP conferred no protection against infertility resulting from direct inoculation of chlamydiae into the oviducts.

Animals↗
PubMed1991

Sequence of the cloned pyr4 gene of Trichoderma reesei and its use as a homologous selectable marker for transformation.

We have cloned and sequenced the Trichoderma reesei pyr4 gene encoding orotidine-5'-monophosphate decarboxylase. Comparison of this sequence with that of the equivalent gene from other filamentous fungi suggests that T. reesei is closely related to Cephalosporium acremonium and Neurospora crassa. The cloned pyr4 gene has been used as a homologous selectable marker for transformation of T. reesei. The majority of transformants obtained with circular plasmid were mitotically unstable and contained non-integrated plasmid molecules, sometimes in addition to plasmid integrated in the genome, Linearization of plasmid prior to transformation decreased the transformation frequency but increased the proportion of stable transformation obtained.

Amino Acid Sequence↗
PubMed1991

Preliminary clinical trials of a computer-based cardiac arrest alarm.

The use of a high reliability cardiac arrest alarm utilising the continuously monitored values of patient heart rate and mean arterial blood pressure is described, based on a sample of 167 patients monitored for a total of 5116 h. The analogue heart rate and mean blood pressure signals are sampled at 1 s intervals, and a smoothing algorithm is applied to each of the resulting series which rejects artefacts, and identifies slope and step changes in each. Certain combinations of events in the 2 series, occurring within a preset time window, determine whether a cardiac arrest alarm or warning signal should be activated by the system. A total of 30 acute events occurring in 14 patients during the course of the study were each identified within 10 s. No cardiac arrest event was misdiagnosed by the algorithm during the period of the study. The algorithm also generates warnings which may have predictive value, and which will be the subject of further research. A final false alarm rate of about 1/200 h of monitoring was observed in adults (1/50 h in children), with evidence that these rates could be substantially improved.

Algorithms↗
PubMed1991

Persistently low natural killer cell activity and circulating levels of plasma beta endorphin: risk factors for infectious disease.

Beta endorphin levels were quantitated in plasma samples obtained from normal subjects (n = 81, 37% males and 63% females, age range 18-45 years) as a component of a prospective study examining the relationship of illness morbidity to natural killer cell activity and psychological indices of stress. The present study was designed to test whether beta endorphin levels contributed additionally to the explanation of illness outcome variance. In the larger study, persistently low NK (LNK) activity was associated prospectively with higher illness morbidity. The findings reported here suggest that the observed LNK activity might be affected by circulating levels of plasma beta endorphin, as lower endorphin levels predicted the LNK pattern, which in turn, predicted higher illness morbidity.

Adolescent↗
PubMed1991

Commercial levels of chymosin production by Aspergillus.

We have increased the production of bovine chymosin in Aspergillus niger var. awamori to more than one gram per liter of secreted authentic enzyme by combining a mutagenesis protocol with a novel robotic screening program. Analysis of the superior chymosin producing strains indicated that they have enhanced capabilities to secrete extracellular proteins.

Aspergillus niger↗
PubMed1991

The use of the Lanthony New Color Test in determining the effects of aging on color vision.

The primary purpose of this study was to collect data on the loss of color vision as a function of age. The Lanthony New Color Test (NCT), which measures acquired losses of color vision in the dimensions of hue, saturation, and brightness, was used to compile data on 68 subjects. The minimum number of subjects were 10 per decade from age 30 to 90 years. An age gradient of selective loss of discrimination of saturation beginning at age 50 was demonstrated, with rapid change noted after age 60. Similar findings were seen for hue but were not evident for brightness. By age 70, a neutral zone emerged at blue/purple, Munsell chroma level 2. The instrument was shown to be reliable and valid in comparison to the Farnsworth Dichotomous Panel D.15. It is seen that this information will provide a basis for planning safer, more functional environments for elderly people.

Adult↗